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1 Article available at or DESCRIPTION OF HAEMONCHUS PLACEI (place, 1893) (nematoda, TRICHOSTRONGYLIDAE, HAEMONCHINAE), IDENTIFICATION AND INTRA-SPECIFIC MORPHOLOGIC VARIABILITY GIUDICI C.J.*, CABARET J.* & DURETTE-DESSET M.C.** Résumé : HAEMONCHUS PLACEI (PLACE, 1893) (NEMATODA, TRICHOSTRONGYLIDAE, HAEMONCHINAE), DESCRIPTION ET VARIABILITÉ MORPHOLOGIQUE INTRA-SPÉCIFIQUE Summary: Haemonchus placei in cattle has never been completely described, possibly due to great morphological similarity with small ruminants Haemonchus contortus. It is newly described on Haemonchus placei chez les bovins n'a jamais été complètement one isolate from Atgentina. It has cleat distinct morphological décrit, sans doute en raison de grandes similitudes features from sheep and goats Haemonchus contortus and presents morphologiques avec Haemonchus contortus des petits ruminants. only two female morphotypes (linguiform and knobbed) instead of Il est ici décrit chez un isolât d'origine argentine. Il a des traits three recorded in H. contortus. A key is proposed to identify morphologiques différents de ceux a"h. contortus parasite d'ovins females. Female as well as male Haemonchus placei from New et de caprins ; seuls deux morphotypes femelles (linguiforme et World (Argentina, Mexico, USA) are morphologically different boutonné) sont recensés au lieu de trois chez H. contortus. Une from those of Old World (Africa: Burkina-Faso, Mauritania and clé est proposée pour la diagnose des femelles. Les mâles ainsi Ivory Coast) or Australia, possibly due to local evolution since their que les femelles sont morphométriquement différents entre le introduction several centuries ago from Africa or India. We Nouveau Monde (Argentine, Mexique et USA) et le Vieux Monde propose to differentiate three sub-species, H. placei placei in (Afrique: Burkina-Faso, Côte-d'lvoire et Mauritanie) et l'australie. Australia, H. placei africanus in western Africa and H. placei Nous proposons d'en faire trois sous-espèces, H. placei placei argentinensis in the New World. pour l'australie, H. placei africanus en Afrique de l'ouest et H. placei argentinensis pour le nouveau Monde. Ces différences KEY WORDS : Haemonchus placei, description, Haemonchus contortus, entre les trois sous-espèces sont probablement le résultat identification, morphologic variability, subspecies. d'adaptations locales d'h.placei placei, après son introduction il y a plusieurs siècles de l'afrique ou de l'inde. MOTS CLES : Haemonchus placei, description, Haemonchus contortus, diagnose, variabilité morphologique, sous-espèces. INTRODUCTION The abomasal worms Haemonchus placei and Haemonchus contortus, are important parasites of domestic ruminants world-wide. Haemonchus placei infects primarily cattle, whereas H. contortus is mainly a parasite of sheep, but the two species are sympatric in several parts of the world. The absence of strict specificity (Jacquiet et al., 1998) indicates need for ways to identify these two species as they might be found together in small ruminants or cattle. Haemonchus contortus was described by Rudolphi (1803) from ovine. Place (1893) named the strongylid-like nematodes he found in the abomasum * INRA, Station de Pathologie Aviaire et de Parasitologie, Nouzilly, France. ** MNHN, Laboratoire de Biologie parasitaire, 61, rue Buffon, Paris Cedex 05, France. Correspondence: J. Cabaret. cabaret@tours.inra.fr of calves suffering from anemic diarrea as Strongylus placei in Southern Australia (Adelaide). Ransom (1911) changed the name of Strongylus placei to Haemonchus placei. Later, Gibbons (1979) regarded this species as a synonym of H. contortus adding to the confusion. Numerous investigations have differentiated the two species. Bremner (1955, 1956) examined the ovine and bovine species of Haemonchus spp. and found that the chromosome number for both was 2 n = 11 in the male and 2n = 12 in the female; he also reported that in the sheep species all the chromosomes were of similar size, whereas in the cattle species the male had two very large chromosomes. Molecular evidence likewise suggests that H. placei and H. contortus are distinct species (Christensen etal, 1994; Zarlenga etal, 1994; Stevenson etal, 1995; Blouin etal., 1997). Interbreeding experiments (Le Jambre, 1979) have demonstrated that they are distinct species. Roberts et al. (1954), Lichtenfels et al. (1988, 1994) found that cattle and sheep Haemonchus spp. were different in the mean length of the spicules and the position of the barbs on these 333

2 GIUDICI C.J., CABARET J. & DURETTE-DESSET M.C spicules. Jacquiet et al. (1997, 1998) differentiated H. placei and H. contortus using discriminant functions based on spicules length. The percentage of body length covered by synlophe (pattern of surface cuticular ridges) was a criterion used by Lichtenfels et al. (1986, 1994). Three main variants of Haemonchus spp. vulval types have been recorded, i.e., those with a linguiform process, those with a knoblike projection, and those with no vulval projection (Chitwood, 1957). Our own unpublished data (morphological and molecular) in H. placei showed that only two types of females are found: linguiform and knobbed, which agrees with the proportions of morphotypes in natural infections already published (Roberts et al. 1954; Tod, 1965). The original description of Place (1893) in Australia was very limited, and no type specimens were deposited, and later, further additional characters were mentioned only to identify H. placei from H. contortus males or females, but no full description of H. placei is available. This is the first aim of the paper. The other objective is to study morphometric variability between Australian, African and New World H. placei, as all cattle has been introduced from Africa to the New World centuries ago (Curasson, 1934) or from South Africa or India to Australia in the last century. MATERIALS AND METHODS HAEMONCHUS PLACEI identification Males The available discriminant function for males based on spicule morphology was used (Jacquiet et al, 1997) as well as the length of body covered with synlophe (Lichtenfels et al., 1994) which extended from 38 to 46 % (Argentina I) corresponding typically to H. placei. Females Three parameters were used to differentiate females of Haemonchus placei from those of Haemonchus contortus: the distance of anus to tail's tip, the width of tail and the proportion of the nematode body length covered by synlophe. The measurements were performed on four isolates (20 females for each one) of Haemonchus contortus: sheep isolate Zaire (Bunia, Ituri), sheep isolate France (Toulouse), goat isolate Caribbean (Guadeloupe), sheep and goat isolate Malaysia (Selangor), and seven isolates of Haemonchus placei (10 females for each one of biomedical study). Five Haemonchus similis (cattle isolate Martinique) were measured as in practice they might be found commonly in cattle. Analysis of variance (Anova) and discriminant analysis was performed using the Stat-Itcf program (1988). Description The worms were collected from the abomasum of two Bos taurus. The redescription was done on 10 males, 10 linguiform females, and 10 knob females of isolate Argentina I from the locality of Pergamino (Buenos- Aires Province) (Museum National d'histoire Naturelle, France, accession number: MNHN 170 MQ). The measurements are given in micrometers unless stated otherwise, with the ranges in parentheses. The nomenclature used for the study of the caudal bursa and synlophe is that of Durette-Desset & Chabaud (1981) and Durette-Desset (1985), respectively. The sublateral hypodermal chords (Lichtenfels & Wergin, 1994) and bilateral perivulvar cutilar pores (Lichtenfels et al, 1995) were not included as they are difficult to see and as they did not discriminate between Haemonchinae species. BlOMETRIC STUDIES Each isolate was studied on specimens collected in one or several bovines. Whole specimens were studied in temporary mounts cleared in phenol-alcohol (80 parts melted phenol crystals and 20 parts absolute alcohol). At least 10 males of every population of putative Haemonchus placei were differentiated of Haemonchus contortus using the discriminant function of Jacquiet et al. (1997, 1998): the studied specimens were thus unequivocally ascribed to H. placei. This was further evaluated in two serial laboratory sheep passages: all male specimens were evidently H. placei based on the same criteria. The parasites of eight cattle isolates were obtained from Pergamino (Argentina, Buenos Aires) isolate Argentina I; Las Parejas (Argentina, Santa Fe) isolate Argentina II; Merida (Yucatan, Mexico) isolate Mexico I; Baton Rouge (United States, Louisiana), isolate USA I; Toumodi (Ivory Coast), isolate Ivory Coast; Bobo-Dioulasso (Burkina Faso), isolate Burkina Faso; Nouakchott (Mauritania) isolate Mauritania, and South-Queensland (Australia) isolate Australia. Larvae from several cattle isolates were passaged once (Mauritania, Burkina Fast;, Argentina II) or during many years (Australia isolate) in sheep. Eight morphological features were measured for 10 males in each of the eight isolates, i.e., body length, body width, oesophagus length, distance from head to cervical papillae, length of spicule, distance from hook to tip of right and left spicules, and gubernaculum length. The following eight measurements were recorded for 10 females in each of the eight isolates, i.e., body length, body width, distance vulva-tail, oesophagus length, distance from head to cervical papillae, synlophe length, distance from anus to posterior extremity and width of tail. All the measurements were made using standard light microscopy and a semi-auto- 334

3 DESCRIPTION OF HAEMONCHUS PLACEI matic image analyser. The proportion of the nematode body length covered by synlophe was made using interference-contrast light microscopy. Discriminant analyses were performed on male and female measures using the Stat-Itcf program (Stat-Itcf, 1988). RESULTS HAEMONCHUS PLACEI IDENTIFICATION USING DISCRIMINANT Ft 'NOTIONS Males Most isolates, sheep H. contortus from Guadeloupe (French West Indies; n = 20), South of France (Toulouse; n = 5), Malaysia {n = 20) and Congo-Zaire (n = 20) and cattle H. placet from Argentina I {n = 40), Argentina II (n = 10), Burkina-Faso (n = 20), or H. similis from Martinique (w = 50) were all correctly identified. Ivory Coast, Mexico, and USA I cattle isolates were repectively constituted of H. placet (20, 80 and 80 %) and H. similis {SO, 20 and 20 %). The Mauritanian cattle isolate was composed of H. placei (90 %) and H. contortus (10 %). Females The synlophe length (Sy), distance anus to tail (Dt), and tail width (Tw) (Table I) were used to establish a discriminant function, as no single measure was able to discriminate well enough between the three species. Using only Sy, there was 15 % of missclassified, mostly H. placei identified as H. similis and conversely using Dt alone, 15 % were missclassified (mostly H. placei identified as H. contortus or vice versa). The first two axes (Yl and Y2) in discriminant function, which are linear combinations of Sy, Dt and Tw, were significant (P < 0.01). The pseudo-f value of Yl (341) was higher than F from any measure taken alone, which indicates that linear combination of measures was more efficient in discriminating species. The Sy (Anova: F = 319 for 2 and 152 degree of freedom) and Dt (Anova: F = 141 for df = 2, 152) were significant whereas Tw was not significantly different between the three species. Only one worm out of 155 was missclasified (one H. placei classified as H. contortus). The discriminant functions are: Yl = x Sy x Dt x Tw Y2 = x Sy x Dt x Tw The following identification key was established on the basis of discriminant analysis: 1 (2) Y2 < H. similis 2 (3) Y2 > H. placei or H. contortus 3-1 Yl > 0 H. placei 3-2 Yl < 0 H. contortus Synlophe Distance Tail Species length anus-tail width H. p. Argentina I 41* (3)" 36-46"* (63) (11) H. p. Argentina II (5) (59) (18) H. p. Mexico I 37 (2) 581 (47) 103 (9) H. p. USA I (3) (44) (15) H. p. Burkina Faso (7) (38) (6) H. p. Mauritania (2) (33) (14) H. p. Ivory Coast (3) (60) (33) H. c. Zaire 57 (4) 421 (42) 106 (10) H. c. France 57 (3) 445 (40) 144 (192) H. c. Caribbea (3) (37) (13) H. c. Malaysia (3) (42) (11) H. similis Carribea 48 (4) 213 (24) 72 (7) H. p. = H. placei. H. c. = H. contortus. * mean in um, ** standard-deviation (in parentheses) and *** range. Table I. - Female measures needed for identification of Haemonchus placei. If. contortus and H. similis. DESCRIPTION OF HAEMONCHUS PLACEI ARGENTTNENSIS N. SUB-SPECIES Body filiform. Head without cephalic vesicle, with a buccal cavity with a lancet-shaped, dorsal neodont formation (Figs. IB, C). In apical view, buccal aperture hexagonal ring, 2 small amphids, 6 externo-labial papillae and 4 cephalic papillae. Presence of 4 cephalic plates, crescent-shaped, situated behind median externo-labial papillae (Fig. ID). Synlophe of type I, present only in anterior part of body, in various proportions: 41 ( ) percent; about 36 ridges in male, 34 in females at oesophagealintestinal level, of more or less equal height and orientated perpendicularly to body surface (Fig. IE). Excretory pore situated just posterior to nerve ring. Hook-shaped deirids, posterior to excretory pore, 335

4 GIUDICI C.J., CABARET J. & DURETTE-DESSET M.C Fig Haemoncbusplacei argentinensis n. sub-species in Bos taurus. Female. A, anterior extremity, ventral view. B-D, head, B, ventral view, C, right lateral view, D, apical view. E, synlophe, transverse section of the body, at the esophago-intestinal junction. F, detail of the nerve ring, excretory pore and deirids, ventral view. G, tail, ventral view. H-I, vulvar region. H, linguiform process and ovejector, right lateral view, I, knob process; r: right side. A, G, H: 200 pm; B-D: 20 urn; E: 50 pm; I, J: 100 pm. 336

5 Fig Haemonchus placet argentinensis n. sub-species in Bos taurus. Male. A, caudal bursa, ventral view. B, genital cone, papillae 0 and 7, dorsal view. C, genital cone, ventral view. D, gubernaculum, ventral view. E-F, spicules, ventral view, E, right spicule, F, left spicule. A, 200 urn; B, C: 50 um; D-F: 100 um. The dorsal rays were coded from 2 to

6 situated very anteriorly, in the first quarter of oesophagus length (Fig. 1A, F). Male: Length 15.2 ( ) mm, width at mid-body 167 ( ), deirids 411 ( ) from cephalic extremity. Oesophagus 1,595 ( ) long. Caudal bursa with ornate ventral surface mainly between extremities of rays 2 to 8, composed of 2 large lateral lobes and a reduced dorsal lobe situated on left (Fig. 2A). Ray 2 and 3 with very long, common stem, curved ventrally with extremities separated from each other. Ray 4 straight, extremities almost equidistant from those of ray 3 and 5. Ray 5 and 6 parallel with extremities curved dorsally. Ray 8 straight and thin. Dorsal ray stout, divided half-way down in 2 branches, each branch divided again into 2, internal (rays 10) longer than external (ray 9). Genital cone prominent, bearing on ventral lip sharp papilla "zero" (Fig. 2C) and 2 curved papillae "7" on dorsal lip (Fig. 2B). Spicules thick, unbranched, subequal, 512 ( ) long. Each spicule had one distally small protuberance (Figs. 2E, F). Distance between right and left protuberance and tip of spicule was 56 (51-61) and 26 (24-28) respectively. Gubernaculum spindle -shaped in ventral view, 253 ( ) long (Fig. 2D). Female: according to presence or absence of a process at vulvar level, the morphometric data are as follows: Females with linguiform process: Length 21 (19-22) mm, width at mid-body 275 ( ). Deirids 471 ( ) from cephalic extremity. Oesophagus 193 ( ). Vulvar opening at 71 %. Thick-walled vagina vera directed forwards parallel to body. Linguiform process, about 400 um, situated above the vulvar opening (Fig. 1H) Rounded tail, without spine, 599 ( ) long and 131 ( ) wide at it is base (Fig. 1G). Females with knob process : Length 20 (19-21) mm, width at mid-body 273 ( ). Deirids 457 ( ) from cephalic extremity. Oesophagus 165 ( ) (Fig. 1A). Vulvar opening at 76 %. Thick-walled vagina vera directed forwards obliquely to body. Knob process, about 150 x 150 um, situated on left side on body (Fig. II). Rounded tail, without spine, 561 ( ) long and 122 ( ) wide at its base. INTRA-SPECIFIC VARIABILITY OF H. PLACEI ISOLATES The different isolates were slightly different according to the morphological characteristics (Tables II and III). All the characters were significant using analysis of variance (P < 0.01). The identification based on eight morphometries characters using discriminant analysis was able to classify correctly only 72 % and 74 % of males and females into the right isolate, respectively. Conversely, three groups were clearly delineated (95 % of males and 85 % of females were well classified) in this analysis: male (79 % of inertia in the significant two axes) or female (75 % of inertia in the significant two axes) H. placet from Australia, Africa and America Oesophagus Cervical Length Right hook to Left hook to Guberna Isolates Body length Body width length deirids* of spicule tip of spicule tip of spicule culum Argentina I 15, , (496) (16) (139) (27) (23) (6) (3) (17) 14,276-15, ,393-1, Argentina II 16, , (1,357) (7) (53) (22) (19) (4) (3) (10) 13,700-17, ,464-1, Mexico I 14, , (1,465) (12) (112) (42) (17) (3) (2) (11) 11,400-16, ,331-1, USA I 15, , (1,006) (17) (78) (3D (16) (2) (3) (16) 14,000-17, ,377-1, Burkina 13, , (567) (8) (73) (8) (15) (2) (3) (10) 12,500-13, ,234-1, Mauritania 14, , (497) (16) (67) (16) (10) (4) (3) (8) 13,200-14, ,266-1, Ivory Coast 13, , (1,697) (17) (99) (14) (19) (3) (3) (18) 10,400-15, ,229-1, Australia 17, , (1,306) (7) (81) (3D (14) (3) (2) (10) 14,709-19, ,609-1, * Distance from head to cervical deirids. Table II. - Male morphometric characteristics (mean in um, standard-deviation and range) of eight isolates of Haemonchusplacei. 338

7 Distance Oesophagus Cervical Synlophe Distance Tail Isolates Body length Body width vulva-tail length deirids* length" anus-tail width Argentina I 18, ,148 1, (1,726) (12) (460) (57) (26) (3) (63) (11) 15,427-21, ,423-3,760 1,589-1, Argentina II 23, ,950 1, (1,999) (23) (334) (136) (35) (5) (59) (18) 20,100-26, ,500-4,600 1,467-1, Mexico I 19, ,520 1, (1,504) (14) 545 (128) (32) (2) (47) (9) 18,000-21, ,000-4,800 1,337-1, USA I 21, ,550 1, (971) (18) (422) (93) (28) (3) (44) (15) 19,800-22, ,700-4,100 1,481-1, Burkina 15, ,750 1, (1,641) (12) (127) (67) (20) (7) (38) (6) 12,800-18, ,500-3,000 1,276-1, Mauritania 18, ,410 1, (867) (14) (331) (82) (15) (2) (33) (14) 17,300-19, ,700-3,900 1,329-1, Ivory Coast 19, ,280 1, (2,139) (25) (442) (104) (25) (3) (60) (33) 17,000-22, ,500-3,900 1,440-1, Australia 22, ,342 1, (2,745) (9) (260) (86) (24) (2) (58) (9) 14,771-24, ,816-4,710 1,571-1, * Distance from head to cervical deirids. ** Percentage of synlophe length on the body length. Table III. - Female morphometric characteristics (mean in um and standard-deviation) of eight isolates of Haemonchusplacet. were different (Fig. 3). Only one isolate from Australia was studied and we focused on New World and African data where several isolates were studied. H. placet found in New World (North and South America) and Old World (Africa) were different when their morphometries was compared (Figs. 4 and 5): 94 % of males and 89 % of females were well identified to their African or American origin. DISCUSSION Species of Haemonchus have veterinary significance and it is important to have accurate species identification. There are nine or ten species and five sub-species of which four are from domestic ruminants (H. longistipes, H. contortus, H. similis and H. placet) (Gibbons, 1979). The differentiation of males by measurement of spicules (Jacquiet et al, 1997) was a simple good criteria for individual identification between Haemonchus contortus and Haemonchus placet, in our isolates. The differentiation of females based on length of synlophe, width and length of tail was completely efficient for the different isolates. Morphometries of males and females were related to their Australian, African or New World location. There has been introduction of probably infected cattle in the last four centuries in the Carribea either from Africa (Curasson, 1934) or India, where H. placet is common. Fig Male and female morphological differences between African, American and Australian isolates using discriminant analysis. Only the gravity centers of American (square), African (circle) and Australian (triangle) isolates are shown. 339

8 Fig Male morphological differences between African and American Haemonchus placet isolates using discriminant analysis. The isolates were coded as Argentina I -1, Argentina II -2, Mexico I -3, USA I -4, Burkina Fasso -5, Mauritania -6, and Ivory Coast -7. O = African isolates. = American isolates; shaded circle and square correspond to gravity centers of each isolate. Fig Female morphological differences between African and American Haemonchus placet isolates using discriminant analysis. The isolates were coded as Argentina I -1, Argentina II -2, Mexico I -3, USA I -4, Burkina Fasso -5, Mauritania -6, and Ivory Coast -7. O = African isolates. = American isolates; shaded circle and square correspond to gravity centers of each isolate. 340

9 Further introductions of cattle from the Carribean to Central or South America did occur which might explain the presence of H. placei in South America. The origin of H. placet in Australia might relate to Indonesian introductions (Lejambre, personnal communication, 1999). The different origin H. placet in America evolved for several centuries (probably for African introduced H. placet) or less than a century (H. placet from massive Indian Bos indicus importation at the very beginning of this century). At that stage, we suggest that the isolate from Australia should be considered as a subspecies called H. placet placet whereas the subspecies found in America and Africa should be called H. placet argentinensis n. sub-species and H. placet africanus n. sub-species, respectively. More detailed studies using genetic markers on a larger number of isolates could eventually trace the migrations of H. placet which could explain the differences between African, Australian and American isolates. Only one isolate from Australia (repeatedly bred for years in sheep) was used to characterise the continent and cattle isolates from several regions should be studied to confirm the particularities of the Australian isolate we described. The existence of several subspecies tends to indicate that H. placei might evoluate relatively rapidly. This would not be the first case rapid evolution as it has been recorded in another trichostrongylid, Heligmosomoides polygyrus, after introduction from Europe to North America at the colonizing period. In less than five centuries, differences in morphological features (Durette-Desset et al., 1972), hosts preferences (Quinnel etal, 1991; N'Zobadila, 1994), and biological characteristics or genetic markers (N'Zobadila, 1994) were evidenced in H. polygyrus and they were separated into H. p. polygyrus (Europe) and H. p. bakeri (North America). ACKNOWLEDGEMENTS We gratefully acknowledge the financial help of FOMEC and the Veterinary Faculty of Casilda (Argentina). Several Haemonchus placei isolates were kindly provided by Gerardo Ardusso (Argentina), Jorge Dominguez (Mexico), John Williams (USA, Florida), Josef Zinnstag and Clarisse Kornoir (Ivory Coast), Lamine Ouattara (Burkina-Faso), Jean-Yves Cholet and Philippe Jacquiet (Mauritania) and Leo Le Jambre (Australia). We are also grateful to Pierre Dorny (Malaysia), Philippe Dorchies (France) and Gilles Aumont (French West Indies) for Haemonchus contortus and Lucas Gruner (France) for Haemonchus similis samples. REFERENCES BLOWN M. 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